Distribution of sodium transporters and aquaporin-1 in the human choroid plexus

The choroid plexus epithelium secretes electrolytes and fluid in the brain ventricular lumen at high rates. Several channels and ion carriers have been identified as likely mediators of this transport in rodent choroid plexus. This study aimed to map several of these proteins to the human choroid plexus. Immunoperoxidase-histochemistry was employed to determine the cellular and subcellular localization of the proteins. The water channel, aquaporin (AQP) 1, was predominantly situated in the apical plasma membrane domain, although distinct basolateral and endothelial immunoreactivity was also observed. The Na⁺-K⁺-ATPase ₁-subunit was exclusively localized apically in the human choroid plexus epithelial cells. Immunoreactivity for the Na⁺-K⁺-2Cl^– cotransporter, NKCC1, was likewise confined to the apical plasma membrane domain of the epithelium. The Cl^–/HCO₃^– exchanger, AE2, was localized basolaterally, as was the Na⁺-dependent Cl^–/HCO₃^– exchanger, NCBE, and the electroneutral Na⁺-HCO₃^– cotransporter, NBCn1. No immunoreactivity was found toward the Na⁺-dependent acid/base transporters NHE1 or NBCe2. Hence, the human choroid plexus epithelium displays an almost identical distribution pattern of water channels and Na⁺ transporters as the rat and mouse choroid plexus. This general cross species pattern suggests central roles for these transporters in choroid plexus functions such as cerebrospinal fluid production. immunohistochemistry; metabolism; cerebrospinal fluid secretion     

Nongenomic regulation by aldosterone of the epithelial NHE3 Na(+)/H(+) exchanger

The relevance of nongenomic pathways to regulation of epithelial function by aldosterone is poorly understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO₃^– absorption in the renal medullary thick ascending limb (MTAL) through a nongenomic pathway. Here, we examined the transport mechanism(s) responsible for this regulation, focusing on Na⁺/H⁺ exchangers (NHE). In the MTAL, apical NHE3 mediates H⁺ secretion necessary for HCO₃^– absorption; basolateral NHE1 influences HCO₃^– absorption by regulating apical NHE3 activity. In microperfused rat MTALs, the addition of 1 nM aldosterone rapidly decreased HCO₃^– absorption by 30%. This inhibition was unaffected by three maneuvers that inhibit basolateral Na⁺/H⁺ exchange and was preserved in MTALs from NHE1 knockout mice, ruling out the involvement of NHE1. In contrast, exposure to aldosterone for 15 min caused a 30% decrease in apical Na⁺/H⁺ exchange activity over the intracellular pH range from 6.5 to 7.7, due to a decrease in V_max. Inhibition of HCO₃^– absorption by aldosterone was not affected by 0.1 mM lumen Zn²⁺ or 1 mM lumen DIDS, arguing against the involvement of an apical H⁺ conductance or apical K⁺-HCO₃^– cotransport. These results demonstrate that aldosterone inhibits HCO₃^– absorption in the MTAL through inhibition of apical NHE3, and identify NHE3 as a target for nongenomic regulation by aldosterone. Aldosterone may influence a broad range of epithelial transport functions important for extracellular fluid volume and acid-base homeostasis through direct regulation of this exchanger. thick ascending limb; acid-base transport; epithelial Na⁺ transport; kidney     

Fluid secretion and the Na+-K+-2Cl- cotransporter in mouse exorbital lacrimal gland

We have previously suggested that fluid flow in the mouse exorbital lacrimal gland is driven by the opening of apical Cl^– and K⁺ channels. These ions move into the lumen of the gland and water follows by osmosis. In many tissues, the Na⁺-K⁺-2Cl^– cotransporter (NKCC1) replaces the Cl^– and K⁺ ions that move into the lumen. We hypothesize that mouse exorbital lacrimal glands would have NKCC1 cotransporters and that they would be important in fluid transport by this gland. We used immunocytochemistry to localize NKCC1-like immunoreactivity to the membranes of the acinar cells as well as to the basolateral membranes of the duct cells. We developed a method to measure tear flow and its composition from mouse glands in situ. Stimulation with the acetylcholine agonist carbachol produced a peak flow followed by a plateau. Ion concentration measurements of this stimulated fluid showed it was high in K⁺ and Cl^–. Treatment of the gland with furosemide, a blocker of the NKCC1 cotransporter, reduced the plateau phase of fluid flow by 30%. Isolated cells exposed to a hypertonic shock shrank by 20% and then showed a regulatory volume increase (RVI). Both the RVI and swelling were blocked by treatment with furosemide. Cells isolated from these glands shrink by 10% in the presence of carbachol. Blocking NKCC1 with furosemide reduced the amount of shrinkage by 50%. These data suggest that NKCC1 plays an important role in fluid secretion by the exorbital gland of mice.     

Expression and localization of rat NBC4c in liver and renal uroepithelium

Previous studies provided functional evidence for electrogenic Na⁺-HCO₃^– cotransport in hepatocytes and in intrahepatic bile duct cholangiocytes. The molecular identity of the transporters mediating electrogenic sodium-bicarbonate cotransport in the liver is currently unknown. Of the known electrogenic Na⁺-HCO₃^– cotransporters (NBC1 and NBC4), we previously showed that NBC4 mRNA is highly expressed in the liver. In the present study, we performed RT-PCR, immunoblotting, and immunohistochemistry to characterize the expression pattern of NBC4 in rat liver and kidney. For immunodetection, a polyclonal antibody against rat NBC4 was generated and affinity purified. Of the known human NBC4 variants, only the rat NBC4c ortholog was detected by RT-PCR in rat liver, and the molecular mass of the NBC4c protein was 145 kDa. NBC4c protein was expressed in hepatocytes and in the cholangiocytes lining the intrahepatic bile ducts. In hepatocytes, NBC4c was localized to the basolateral plasma membrane, whereas intrahepatic cholangiocytes stained apically. The NBC1 electrogenic sodium cotransporter variants kNBC1 and pNBC1 were not detected by immunoblotting and immunohistochemistry in rat liver. The pattern of localization of NBC4c in the liver suggests that the cotransporter plays a role in mediating Na⁺-HCO₃^– cotransport in hepatocytes and intrahepatic cholangiocytes. Unlike the liver, the rat kidney expressed electrogenic sodium-bicarbonate cotransporter proteins kNBC1 and NBC4c. In kidney, NBC4c also had a molecular mass of 145 kDa and was immunolocalized to uroepithelial cells lining the renal pelvis, where the cotransporter may play an important role in protecting the renal parenchyma from alterations in urine pH. bicarbonate; transport; electrogenic     

Dynamic regulation of Na(+)-K(+)-2Cl(-) cotransporter surface expression by PKC-{epsilon} in Cl(-)--secretory epithelia

In secretory epithelia, activation of PKC by phorbol ester and carbachol negatively regulates Cl^– secretion, the transport event of secretory diarrhea. Previous studies have implicated the basolateral Na⁺-K⁺-2Cl^– cotransporter (NKCC1) as a target of PKC-dependent inhibition of Cl^– secretion. In the present study, we examined the regulation of surface expression of NKCC1 in response to the activation of PKC. Treatment of confluent T84 intestinal epithelial cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) reduced the amount of NKCC1 accessible to basolateral surface biotinylation. Loss of cell surface NKCC1 was due to internalization as shown by 1) the resistance of biotinylated NKCC1 to surface biotin stripping after incubation with PMA and 2) indirect immunofluorescent labeling. PMA-induced internalization of NKCC1 is dependent on the -isoform of PKC as determined on the basis of sensitivity to a panel of PKC inhibitors. The effect of PMA on surface expression of NKCC1 was specific because PMA did not significantly alter the amount of Na⁺-K⁺-ATPase or E-cadherin available for surface biotinylation. After extended PMA exposure (>2 h), NKCC1 became degraded in a proteasome-dependent fashion. Like PMA, carbachol reduced the amount of NKCC1 accessible to basolateral surface biotinylation in a PKC--dependent manner. However, long-term exposure to carbachol did not result in degradation of NKCC1; rather, NKCC1 that was internalized after exposure to carbachol was recycled back to the cell membrane. PKC--dependent alteration of NKCC1 surface expression represents a novel mechanism for regulating Cl^– secretion. endocytosis; recycling; ion transporters     

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport     

Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4

In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na⁺-K⁺-2Cl^– cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K⁺-Cl^– cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2. bumetanide; Na⁺-K⁺-2Cl^– cotransporter; K⁺-Cl^– cotransporter; Xenopus oocytes     

Moderate-to-severe ischemic conditions increase activity and phosphorylation of the cerebral microvascular endothelial cell Na+-K+-Cl- cotransporter

Brain edema that forms during the early stages of stroke involves increased transport of Na⁺ and Cl^– across an intact blood-brain barrier (BBB). Our previous studies have shown that a luminal BBB Na⁺-K⁺-Cl^– cotransporter is stimulated by conditions present during ischemia and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema formation in the rat middle cerebral artery occlusion model of stroke. The present study focused on investigating the effects of hypoxia, which develops rapidly in the brain during ischemia, on the activity and expression of the BBB Na⁺-K⁺-Cl^– cotransporter, as well as on Na⁺-K⁺-ATPase activity, cell ATP content, and intracellular volume. Cerebral microvascular endothelial cells (CMECs) were assessed for Na⁺-K⁺-Cl^– cotransporter and Na⁺-K⁺-ATPase activities as bumetanide-sensitive and ouabain-sensitive ⁸⁶Rb influxes, respectively. ATP content was assessed by luciferase assay and intracellular volume by [³H]-3-O-methyl-D-glucose and [¹⁴C]-sucrose equilibration. We found that 30-min exposure of CMECs to hypoxia ranging from 7.5% to 0.5% O₂ (vs. 19% normoxic O₂) significantly increased cotransporter activity as did 7.5% or 2% O₂ for up to 2 h. This was not associated with reduction in Na⁺-K⁺-ATPase activity or ATP content. CMEC intracellular volume increased only after 4 to 5 h of hypoxia. Furthermore, glucose and pyruvate deprivation increased cotransporter activity under both normoxic and hypoxic conditions. Finally, we found that hypoxia increased phosphorylation but not abundance of the cotransporter protein. These findings support the hypothesis that hypoxia stimulation of the BBB Na⁺-K⁺-Cl^– cotransporter contributes to ischemia-induced brain edema formation. edema; blood-brain barrier; bumetanide; cell volume     

ATP-sensitive potassium channels mediate hyperosmotic stimulation of NKCC in slow-twitch muscle

In mildly hyperosmotic medium, activation of the Na⁺-K⁺-2Cl^- cotransporter (NKCC) counteracts skeletal muscle cell water loss, and compounds that stimulate protein kinase A (PKA) activity inhibit the activation of the NKCC. The aim of this study was to determine the mechanism for PKA inhibition of NKCC activity in resting skeletal muscle. Incubation of rat slow-twitch soleus and fast-twitch plantaris muscles in isosmotic medium with the PKA inhibitors H-89 and KT-5720 caused activation of the NKCC only in the soleus muscle. NKCC activation caused by PKA inhibition was insensitive to MEK MAPK inhibitors and to insulin but was abolished by the PKA stimulators isoproterenol and forskolin. Furthermore, pinacidil [an ATP-sensitive potassium (K_ATP) channel opener] or inhibition of glycolysis increased NKCC activity in the soleus muscle but not in the plantaris muscle. Preincubation of the soleus muscle with glibenclamide (a K_ATP channel inhibitor) prevented the NKCC activation by hyperosmolarity, PKA inhibition, pinacidil, and glycolysis inhibitors. In contrast, glibenclamide stimulated NKCC activity in the plantaris muscle. In cells stably transfected with the Kir6.2 subunit of the of K_ATP channel, inhibition of glycolysis activated potassium current and NKCC activity. We conclude that activation of K_ATP channels in slow-twitch muscle is necessary for activation of the NKCC and cell volume restoration in hyperosmotic conditions. protein kinase A; glibenclamide; glycolysis; Na⁺-K⁺-2Cl^- cotransporter; Kir6.2     

NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia

Cytoplasmic pH (pH_i) was evaluated duringNa⁺-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pH_i increased by 0.069 ± 0.002 within 150 s after initiation of Na⁺-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na⁺-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa⁺/H⁺ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa⁺-glucose cotransport with an ED₅₀ of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pH_i increases at <500 µM.Na⁺-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH_i increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na⁺-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa⁺-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na⁺ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

     

Stimulation of astrocyte Na+/H+ exchange activity in response to in vitro ischemia depends in part on activation of ERK1/2

We recently reported that Na⁺/H⁺ exchanger isoform 1 (NHE1) activity in astrocytes is stimulated and leads to intracellular Na⁺ loading after oxygen and glucose deprivation (OGD). However, the underlying mechanisms for this stimulation of NHE1 activity and its impact on astrocyte function are unknown. In the present study, we investigated the role of the ERK1/2 pathway in NHE1 activation. NHE1 activity was elevated by 75% in NHE1^+/+ astrocytes after 2-h OGD and 1-h reoxygenation (REOX). The OGD/REOX-mediated stimulation of NHE1 was partially blocked by 30 µM PD-98059. Increased expression of phosphorylated ERK1/2 was detected in NHE1^+/+ astrocytes after OGD/REOX. Moreover, stimulation of NHE1 activity disrupted not only Na⁺ but also Ca²⁺ homeostasis via reverse-mode operation of Na⁺/Ca²⁺ exchange. OGD/REOX led to a 103% increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in NHE1^+/+ astrocytes in the presence of thapsigargin. Inhibition of NHE1 activity with the NHE1 inhibitor HOE-642 decreased OGD/REOX-induced elevation of [Ca²⁺]_i by 73%. To further investigate changes of Ca²⁺ signaling, bradykinin-mediated Ca²⁺ release was evaluated. Bradykinin-mediated intracellular Ca²⁺ transient in NHE1^+/+ astrocytes was increased by 84% after OGD/REOX. However, in NHE1^–/– astrocytes or NHE1^+/+ astrocytes treated with HOE-642, the bradykinin-induced Ca²⁺ release was increased by only 34%. Inhibition of the reverse mode of Na⁺/Ca²⁺ exchange abolished OGD/REOX-mediated Ca²⁺ rise. Together, our data suggest that ERK1/2 is involved in activation of NHE1 in astrocytes after in vitro ischemia. NHE1-mediated Na⁺ accumulation subsequently alters Ca²⁺ homeostasis via Na⁺/Ca²⁺ exchange. intracellular pH; cortical astrocytes; sodium/calcium exchange; intracellular sodium ion     

K+ channel KVLQT1 located in the basolateral membrane of distal colonic epithelium is not essential for activating Cl- secretion

The cellular mechanism for Cl^– and K⁺ secretion in the colonic epithelium requires K⁺ channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the K⁺ channel proteins K_VLQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current (I_sc) and transepithelial conductance (G_t) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin E₂ (PGE₂), and carbachol (CCh). HMR1556 (10 µM), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Brüggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 3831–3837, 2001), partially (50%) inhibited Cl^– secretory I_sc and G_t activated by PGE₂ and CCh in rat colon with an IC₅₀ of 55 nM, but in guinea pig distal colon Cl^– secretory I_sc and G_t were unaltered. EPI-activated K⁺-secretory I_sc and G_t also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 K⁺ channel is not an essential component for producing Cl^– secretion. Other K⁺ channels present in the basolateral membrane presumably must also contribute directly to the K⁺ conductance necessary for K⁺ exit during activation of Cl^– secretion in the colonic mucosa. HMR1556; K⁺ secretion; epinephrine; prostaglandin E₂; cholinergic     

Immunolocalization of Na/K–ATPase and Na/K/2Cl cotransporter in the tubular epithelia of sea snake salt glands

The sublingual salt gland is the primary site of salt excretion in sea snakes; however, little is known about the mechanisms mediating ion excretion. Na⁺/K⁺–ATPase (NKA) and Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) are two proteins known to regulate membrane potential and drive salt secretion in most vertebrate secretory cells. We hypothesized that NKA and NKCC would localize to the basolateral membranes of the principal cells comprising the tubular epithelia of sea snake salt glands. Although there is evidence of NKA activity in salt glands from several species of sea snake, the localization of NKA and NKCC and other potential ion transporters remains unstudied. Using histology and immunohistochemistry, we localized NKA and NKCC in salt glands from three species of laticaudine sea snake: Laticauda semifasciata, L. laticaudata, and L. colubrina. Antibody specificity was confirmed using Western blots. The compound tubular glands of all three species were found to be composed of serous secretory epithelia, and NKA and NKCC were abundant in the basolateral membranes. These results are consistent with the morphology of secretory epithelia found in the rectal salt glands of marine elasmobranchs, the nasal glands of marine birds and the gills of teleost fishes, suggesting a similar function in regulating ion secretion.     

A new model for fish ion regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes)

The ion regulation mechanisms of fishes have been recently studied in zebrafish (Danio rerio), a stenohaline species. However, recent advances using this organism are not necessarily applicable to euryhaline fishes. The euryhaline species medaka (Oryzias latipes), which, like zebrafish, is genetically well categorized and amenable to molecular manipulation, was proposed as an alternative model for studying osmoregulation during acclimation to different salinities. To establish its suitability as an alternative, the present study was conducted to (1) identify different types of ionocytes in the embryonic skin and (2) analyze gene expressions of the transporters during seawater acclimation. Double/triple in situ hybridization and/or immunocytochemistry revealed that freshwater (FW) medaka contain three types of ionocyte: (1) Na⁺/H⁺ exchanger 3 (NHE3) cells with apical NHE3 and basolateral Na⁺-K⁺-2Cl^? cotransporter (NKCC), Na⁺-K⁺-ATPase (NKA) and anion exchanger (AE); (2) Na⁺-Cl^? cotransporter (NCC) cells with apical NCC and basolateral H⁺-ATPase; and (3) epithelial Ca²⁺ channel (ECaC) cells [presumed accessory (AC) cells] with apical ECaC. On the other hand, seawater (SW) medaka has a single predominant ionocyte type, which possesses apical cystic fibrosis transmembrane conductance regulator (CFTR) and NHE3 and basolateral NKCC and NKA and is accompanied by smaller AC cells that express lower levels of basolateral NKA. Reciprocal gene expressions of decreased NHE3, AE, NCC and ECaC and increased CFTR and NKCC in medaka gills during SW were revealed by quantative PCR analysis.     

Cytosolic potassium controls CFTR deactivation in human sweat duct

Absorptive epithelial cells must admit large quantities of salt (NaCl) during the transport process. How these cells avoid swelling to protect functional integrity in the face of massive salt influx is a fundamental, unresolved problem. A special preparation of the human sweat duct provides critical insights into this crucial issue. We now show that negative feedback control of apical salt influx by regulating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel activity is key to this protection. As part of this control process, we report a new physiological role of K⁺ in intracellular signaling and provide the first direct evidence of acute in vivo regulation of CFTR dephosphorylation activity. We show that cytosolic K⁺ concentration ([K⁺]_c) declines as a function of increasing cellular NaCl content at the onset of absorptive activity. Declining [K⁺]_c cause parallel deactivation of CFTR by dephosphorylation, thereby limiting apical influx of Cl^– (and its co-ion Na⁺) until [K⁺]_c is stabilized. We surmise that [K⁺]_c stabilizes when Na⁺ influx decreases to a level equal to its efflux through the basolateral Na⁺-K⁺ pump thereby preventing disruptive changes in cell volume. electrolytes; phosphatases; protein kinase A; cystic fibrosis transmembrane conductance regulator; epithelial Na⁺ channel     

The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia

The human electrogenic renal Na-HCO₃ cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO₃^– from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na⁺ affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules. bicarbonate; intracellular pH; acidbase; SLC4A4; Na⁺-HCO₃ cotransporter 1     

Physiological relevance of cell-specific distribution patterns of CFTR, NKCC1, NBCe1, and NHE3 along the crypt-villus axis in the intestine

We examined the cell-specific subcellular expression patterns for sodium- and potassium-coupled chloride (NaK2Cl) cotransporter 1 (NKCC1), Na(+) bicarbonate cotransporter (NBCe1), cystic fibrosis transmembrane conductance regulator (CFTR), and Na(+)/H(+) exchanger 3 (NHE3) to understand the functional plasticity and synchronization of ion transport functions along the crypt-villus axis and its relevance to intestinal disease. In the unstimulated intestine, all small intestinal villus enterocytes coexpressed apical CFTR and NHE3, basolateral NBCe1, and mostly intracellular NKCC1. All (crypt and villus) goblet cells strongly expressed basolateral NKCC1 (at approximately three-fold higher levels than villus enterocytes), but no CFTR, NBCe1, or NHE3. Lower crypt cells coexpressed apical CFTR and basolateral NKCC1, but no NHE3 or NBCe1 (except NBCe1-expressing proximal colonic crypts). CFTR, NBCe1, and NKCC1 colocalized with markers of early and recycling endosomes, implicating endocytic recycling in cell-specific anion transport. Brunner's glands of the proximal duodenum coexpressed high levels of apical/subapical CFTR and basolateral NKCC1, but very low levels of NBCe1, consistent with secretion of Cl(-)-enriched fluid into the crypt. The cholinergic agonist carbachol rapidly (within 10 min) reduced cell volume along the entire crypt/villus axis and promoted NHE3 internalization into early endosomes. In contrast, carbachol induced membrane recruitment of NKCC1 and CFTR in all crypt and villus enterocytes, NKCC1 in all goblet cells, and NBCe1 in all villus enterocytes. These observations support regulated vesicle traffic in Cl(-) secretion by goblet cells and Cl(-) and HCO(3)(-) secretion by villus enterocytes during the transient phase of cholinergic stimulation. Overall, the carbachol-induced membrane trafficking profile of the four ion transporters supports functional plasticity of the small intestinal villus epithelium that enables it to conduct both absorptive and secretory functions.     

Vasoconstrictors and nitrovasodilators reciprocally regulate the Na+-K+-2Cl- cotransporter in rat aorta

Little is knownabout the function and regulation of theNa⁺-K⁺-2Clcotransporter NKCC1 in vascular smooth muscle. Theactivity of NKCC1 was measured as the bumetanide-sensitive efflux of⁸⁶Rb⁺from intact smooth muscle of the rat aorta. Hypertonic shrinkage (440 mosmol/kgH₂O) rapidlydoubled cotransporter activity, consistent with its volume-regulatoryfunction. NKCC1 was also acutely activated by the vasoconstrictors ANGII (52%), phenylephrine (50%), endothelin (53%), and 30 mM KCl(54%). Both nitric oxide and nitroprusside inhibited basal NKCC1activity (39 and 34%, respectively), and nitroprussidecompletely reversed the stimulation by phenylephrine. Thephosphorylation of NKCC1 was increased by hypertonic shrinkage, phenylephrine, and KCl and was reduced by nitroprusside. The inhibition of NKCC1 significantly reduced the contraction of rat aorta induced byphenylephrine (63% at 10 nM, 26% at 30 nM) but not by KCl. Weconclude that theNa⁺-K⁺-2Clcotransporter in vascular smooth muscle is reciprocally regulated byvasoconstrictors and nitrovasodilators and contributes to smooth musclecontraction, indicating that alterations in NKCC1 could influencevascular smooth muscle tone in vivo.

     

Role of Na+-K+-Cl- cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia

Na⁺-K⁺-Cl^– cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na⁺/Ca²⁺ exchanger (NCX) contribute to intracellular Na⁺ and Ca²⁺ overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca²⁺ (Ca_m²⁺) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca²⁺ (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca²⁺ was reduced by 42% (P < 0.05). Genetic ablation of NKCC1 also reduced Ca_m²⁺ accumulation. Moreover, OGD/REOX in NKCC1^+/+ astrocytes caused dissipation of the mitochondrial membrane potential (_m) to 42 ± 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of _m was attenuated significantly (66 ± 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca²⁺ that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Ca_m²⁺ was accompanied by loss of _m and cytochrome c release in NKCC1^+/+ astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca_m²⁺ homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia. intracellular calcium ion; mitochondrial membrane potential; sodium ion influx; bumetanide; cytochrome c; glial cell death     

Defective coupling of apical PTH receptors to phospholipase C prevents internalization of the Na+-phosphate cotransporter NaPi-IIa in Nherf1-deficient mice

Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na⁺-phosphate cotransporter (NaP_i-IIa) in the brush border membrane (BBM). The activity and localization of NaP_i-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaP_i-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na⁺/H⁺-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaP_i-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaP_i-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1–34) led to internalization of NaP_i-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3–34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of NaP_i-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaP_i-IIa and, specifically, couples the apical PTHR to PLC. phosphate cotransporter; PDZ protein; parathyroid hormone; proximal tubule